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Cannabinoid receptor dependent and independent anti-proliferative effects of omega-3
ethanolamides in androgen receptor positive and negative prostate cancer cell lines.
Iain Brown*, Maria G. Cascio†, Klaus W.J. Wahle*, Reem Smoum**,
Raphael Mechoulam**, Ruth A. Ross†, Roger G. Pertwee†, Steven D. Heys*.
*Translational Medical Sciences, Division of Applied Medicine, School of Medicine and Dentistry; † Neurobiology Research Programme, School of Medical Sciences, University of Aberdeen, Aberdeen, AB25 2ZD, UK, ** Medical Faculty, Hebrew University, Ein Kerem Campus, 91120 Jerusalem, Israel.
Address for correspondence: Dr Iain Brown Division of Applied Medicine University of Aberdeen, Polwarth Building, Aberdeen, AB25 2ZD, UK. Tel: +44 1224 558989 Fax: +44 1224 555199 E-mail: [email protected]
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Abstract
The omega-3 fatty acid ethanolamides, docosahexaenoyl ethanolamide (DHEA) and
eicosapentaenoyl ethanolamide (EPEA), displayed greater anti-proliferative potency than
their parent omega-3 fatty acids, docosahexaenoic acid (DHA) and eicosapentaenoic acid
(EPA), in LNCaP and PC3 prostate cancer cells. DHEA and EPEA activated cannabinoid
CB1 and CB2 receptors in vitro with significant potency, suggesting that they are
endocannabinoids. Both LNCaP and PC3 cells expressed CB1 and CB2 receptors, and the
CB1- and CB2-selective antagonists, AM281, and AM630, administered separately or
together, reduced the anti-proliferative potencies of EPEA and EPA but not of DHEA or
DHA in PC3 cells and of EPA but not of EPEA, DHEA or DHA in LNCaP cells. Even so,
EPEA and EPA may not have inhibited PC3 or LNCaP cell proliferation via cannabinoid
receptors since the anti-proliferative potency of EPEA was well below the potency it
displayed as a CB1 or CB2 receptor agonist. Indeed, these receptors may mediate a
protective effect, since the anti-proliferative potency of DHEA in LNCaP and PC3 cells was
increased by separate or combined administration of AM281 and AM630. The anandamide-
metabolizing enzyme, fatty acid amide hydrolase (FAAH), was highly expressed in LNCaP
but not PC3 cells. Evidence was obtained that FAAH metabolizes EPEA and DHEA and
that the anti-proliferative potencies of these ethanolamides in LNCaP cells can be enhanced
by inhibiting this enzyme. Our findings suggest that the expression of cannabinoid receptors
and of FAAH in some tumour cells could well influence the effectiveness of DHA and EPA
or their ethanolamide derivatives as anticancer agents.
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Introduction Our group, and others, have shown that the omega-3 long chain polyunsaturated fatty acids,
docosahexaenoic acid (DHA;22:6 n-3), and eicosapentaenoic acid (EPA; 20:5(n-3)), elicit
anti-proliferative, anti-cancer effects both in cancer lines in vitro and in animals in vivo (1,
2). There is also evidence, that dietary omega-3 and omega-6 fatty acids can be converted to
their ethanolamide derivatives in situ. Furthermore, the polyunsaturated omega-6 fatty acid
ethanolamide, arachidonoyl ethanolamide (anandamide), is an endocannabinoid since it can
activate cannabinoid CB1 and CB2 receptors when synthesized and released endogenously,
as well as when administered exogenously (3, 4). Endocannabinoids can have anti-cancer
effects, since there is evidence that anandamide can inhibit human breast cancer and prostate
cancer cell proliferation (5, 6).
In this investigation we explored the possibility that the anticancer effects of DHA and EPA
depend, at least in part, on their conversion to their ethanolamides: DHA to
docosahexaenoyl ethanolamide (DHEA), and EPA to eicosapentaenoyl ethanolamide
(EPEA). The difference in the structures of EPEA, DHEA and anandamide can be seen in
Figure 1A. Because there is evidence that anandamide acts through CB1 receptors to inhibit
the proliferation of human breast and prostate cancer cells we tested the hypothesis that
DHA, EPA, DHEA and/or EPEA produce their anticancer effects by activating cannabinoid
receptors in these cells (5, 6). There is evidence that anandamide can be catabolised by fatty
acid amide hydrolase (FAAH), and that inhibition of this enzyme can potentiate some
effects of anandamide (7, 8). Accordingly, we explored the possibility that the anticancer
effects of DHA and EPA and/or of their ethanolamides can be potentiated by inhibiting this
enzyme.
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Our experiments were performed in vitro, mainly with the human prostate cancer cell lines,
LNCaP (androgen sensitive) and PC3 (androgen insensitive). To investigate whether DHEA
or EPEA are cannabinoid CB1 or CB2 receptor agonists, we performed receptor-binding
experiments with mouse whole-brain membranes and with membranes obtained from
Chinese hamster ovary (CHO) cells that had been stably transfected with the human CB2
receptor.
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Materials and methods
Cell lines
Chinese hamster ovary cells (CHO), stably transfected with cDNA encoding human
cannabinoid CB2 receptors, were cultured in Eagle’s medium nutrient mixture F-12 HAM
supplemented with 1 mM L-glutamine, 10% (v:v) foetal bovine serum and 0.6% penicillin-
streptomycin together with G418 (600 μg ml-1). Human prostate cancer cell lines, LNCaP
(androgen sensitive) and PC3 (androgen insensitive), were obtained from the European
Collection of Animal Cell Cultures. These were cultured in RPMI 1640 medium (Sigma
chemical, Dorset, UK) containing 10% (v:v) foetal bovine serum and 1% (v:v) Penicillin-
Streptomycin solution (10,000 units/ml penicillin, 10 mg/ml streptomycin in 0.9% sodium
chloride, Sigma Aldrich, Dorset, UK).
Animals
All animal care and experimental procedures complied with the UK Animals (Scientific
Procedures) Act, 1986 and associated guidelines for the use of experimental animals. MF1
mice aged 6 to 7 weeks and weighing 30 to 35 g were purchased from Harlan UK Ltd.
(Blackthorn, UK). Mice were maintained on a 12/12h light/dark cycle with free access to
food and water. All experiments were performed with tissues obtained from adult male
mice.
Biochemical reagents
Docosahexaenoic acid (DHA;22:6 n-3), and eicosapentaenoic acid (EPA; 20:5(n-3)) were
purchased from Sigma-Aldrich (Dorset, UK). Eicosapentaenoyl ethanolamide (EPEA) and
Docosahexaenoyl ethanolamide (DHEA) were synthesized in Professor Mechoulam’s
laboratory (9). Fatty acids and ethanolamides were each dissolved in 100% ethanol, and
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FAAH inhibitor, JNJ1661010 (Tocris Bioscience, Bristol, UK), and the CB1 selective
antagonist, AM281 (Insight Biotechnology, Middlesex, UK), were dissolved in DMSO, and
all stored at 100 mM stock solutions, -80°C under nitrogen. The CB2 selective antagonist,
AM630 (Tocris Bioscience, Bristol, UK), was stored in DMSO as a 10 mM solution.
Appropriate concentrations were freshly prepared from stock solution using culture medium.
DMSO and ethanol diluent controls were also in. For binding experiments, [3H]CP-55940
(160 Ci mmol-1) and [35S]GTPγS (1250 Ci mmol-1) were obtained from PerkinElmer Life
Sciences Inc. (Boston, MA, USA), GTPγS and adenosine deaminase from Roche Diagnostic
(Indianapolis, IN, USA) and GDP and PMSF from Sigma-Aldrich.
Cell viability assay
A standard 3-[4,-5-dimethylthiazol]-2,-5-diphenyl tetrazolium bromide (MTT) dye reduction
assay was used to assess the cytotoxicity of the respective compounds. Briefly, cells were
plated in a flat bottomed, 96 well plate at seeding densities of 6x103 cells/well for LNCaP
cells and 5x103 cell/well for PC3 cells. Cells were treated the following day with appropriate
agents for 24 hours. Following the incubation period, the MTT solution (5mg/ml in PBS)
was added and incubated for 4 h. The contents of the wells were removed and replaced with
200 µl DMSO to dissolve the MTT formazan crystals. The plates were immediately read at
570 nM in a multiwell plate reader (DynaTech MR5000, DynaTech Laboratories Inc.,
USA).
Membrane preparation
Binding assays with [3H]CP-55940 and with [35S]GTPγS were performed with mouse whole
brain membranes, prepared as described previously, or with CHO-hCB2 cell membranes(10,
11). The hCB2 transfected cells were removed from flasks by scraping and then frozen as a
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pellet at -20 ºC until required(12). Before use in a radioligand binding assays, cells were
defrosted, diluted in Tris-buffer (50 mM Tris-HCl,50 mM Tris-Base) and homogenized.
Protein assays were performed using a Bio-Rad Dc kit (Hercules, CA, USA).
[35S]GTPγS binding assays
Measurement of agonist-stimulated [35S]GTPγS binding to cannabinoid CB1 receptors was
adapted from previously described methods (12, 13) The assays were carried out with
GTPγS binding buffer (50 mM Tris-HCl, 50 mM Tris-Base, 5 mM MgCl2, 1 mM EDTA,
100 mM NaCl, 1 mM dithiothreitol, 0.1% BSA) in the presence of [35S]GTPγS and GDP, in
a final volume of 500 μl. Binding was initiated by the addition of [35S]GTPγS to the wells.
Nonspecific binding was measured in the presence of 30 μM GTPγS. The drugs were
incubated in the assay for 60 min at 30 ºC. The reaction was terminated by a rapid vacuum
filtration method using Tris-binding buffer, as described previously, and the radioactivity
was quantified by liquid scintillation spectrometry. In all the [35S]GTPγS binding assays, we
used 0.1 nM [35S]GTPγS, 30 μM GDP and a protein concentration of 5 μg per well for
mouse brain membranes and 33 μg per well for CHO-hCB2 cell membranes. Additionally,
mouse brain membranes were pre-incubated for 30 min at 30 ºC with 0.5 U ml-1 adenosine
deaminase (200 U ml-1) to remove endogenous adenosine. Compounds were stored at -20 ºC
in oil.
Protein analysis
Cells were homogenised in lysis buffer (20 mM Tris, 0.25 M sucrose, 10 mM EGTA, 2 mM
EDTA, 1 mM sodium orthovanadate, 25 mM sodium β-glycerophosphate, 50 mM sodium
fluoride, pH 7.5). Prior to use, 0.1% (v:v) Protease Inhibitor Cocktail (Sigma, Dorset, UK)
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was added. A total of 20 µg of protein was electrophoresed through a precast 16%
polyacrylamide gel (Invitrogen, Paisley, UK) for 2 hours and the separated proteins were
transferred to nitrocellulose membranes (Biorad, Hertfordshire, UK), then blocked with 5%
(w:v) skimmed milk in tris buffered saline (TBS) with 0.1% (v:v) Tween 20 (TBST
solution) at room temperature and incubated with 1:200 dilution of anti-FAAH antibody,
1:100 CB1 antibody (Calbiochem, UK), or 1:200 CB2 antibody (Insight Biotechnology, UK)
at 4°C overnight. β-actin (1:20,000) was used as an internal loading control to normalize
between lanes during densitometry. Appropriate secondary antibody for FAAH (anti-
mouse) and CB1 and CB2 (anti-goat) were used at a concentration of 1:5000 (Insight
Biotechnology, UK), and incubated at room temperature for 1 hour. Proteins were visualized
using ECL+plusTM chemiluminescent detection kit (Amersham Pharmacia,
Buckinghamshire, UK), according to manufacturer’s instructions and a Fluor S
phosphorimager (Biorad, Hertfordshire, UK). The experiments were performed with
proteins isolated from three independent extractions.
Flow cytometry analysis of the cell cycle
Effects of the fatty acid and ethanolamide treatments on the cell cycle were determined
using the PI staining technique. Briefly, cells were grown in 6 well plates at a density of 6 ×
104 cells per well and treated with appropriate concentrations of reagents (IC50 values) for 24
hours. Cells were collected by trypsinisation, including the floating cells and fixed in ice
cold absolute alcohol and stained with propidium iodide (PI) for 30 min (50 µg/ml PI, 50
µg/ml ribonuclease A and 0.1% v/v triton x-100 in PBS). The percentage of cells in G1, S,
G2, M and sub G1 phases was assessed by flow cytometry using FACSCalibur flow
cytometer and anlaysed by Flowjo software (Teestar Inc, Oregon, USA).
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Flow cytometry analysis of apoptosis by Annexin V staining
Cells were seeded and treated as for cell cycle analysis, and harvested by brief trypsinisation
and washed in cold PBS and then twice in Annexin Binding Buffer (Beckton Dickinson,
Oxford, UK). Cells were resuspended in 100 µl of binding buffer, and 5 ul FITC-Annexin V
solution (Beckton Dickinson, Oxford, UK) was added and cells incubated for 15 minutes in
the dark at room temperature. A further 400 ul of binding buffer was added prior to
analysis, followed by 2 μl of 1mg/ml solution of propidium iodide, where appropriate. A
total of 10,000 cells were counted and anlaysed by Flowjo software (Teestar Inc, Oregon,
USA). Jurkat cells treated with 6 uM camptothecin (Sigma-Aldrich, Dorset, UK) were used
as a positive control of apoptosis to set the compensation and voltages.
Data analysis
Values have been expressed as means and variability as SEM or as 95% confidence limits.
The concentration of the compounds under investigation that produced a 50% displacement
of radioligand from specific binding sites (IC50 values) was calculated using GraphPad
Prism and the corresponding Ki values were calculated using the equation of Cheng and
Prusoff (14). Net agonist stimulated [35S]GTPγS binding values were calculated by
subtracting basal binding values (obtained in the absence of agonist) from agonist-stimulated
values (obtained in the presence of agonist) as detailed elsewhere (10). Values for EC50 (half
of the maximal effective concentration required to elicit a response), maximal effect (Emax)
and SEM or 95% confidence limits of these values have been calculated by nonlinear
regression analysis using the equation for a sigmoid concentration-response curve
(GraphPad Prism). Unpaired Students t-test, or one-way ANOVA (with Tukeys post-hoc
analysis) were used where appropriate for MTT data and flow cytometry data. A value of
p<0.05 was taken as being significant.
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Results
The ethanolamides of EPA and DHA induce cell death in LNCaP and PC3 cells
EPEA was more potent than EPA in inducing cell death in both LNCaP (p<0.01) and PC3
cells (p<0.01). Similarly, DHEA was more potent than DHA in both LNCaP (p<0.01) and
PC3 cells (p<0.05), (Figure 1B and C). Treatment of LNCaP cells with EPA or EPEA did
not result in any significant changes in the G1, S or G2 phases of the cell cycle, (Table 1).
However, DHA did induce a significant decrease in both G2 (p< 0.05) and S phases
(p<0.05), whereas DHEA caused a significant decrease in the G1 phase compared to
untreated cells (p<0.05), and also compared to DHA (p<0.05). Treatment of PC3 cells with
EPA resulted in a significant increase in G1 phase cells (p<0.01), and a significant decrease
in S phase (p<0.01), when compared to untreated cells (Table 1). EPEA treatment did not
significantly alter the PC3 cell cycle in any phase, but did induce a significant decrease in
G1 when compared to its parent EPA counterpart (p<0.01), (see supplemental figure 1A).
DHA elicited a significant decrease in G2 phase PC3 cells (p<0.01) but DHEA showed an
increase in G2 compared to untreated cells, although this was of borderline significance
(p=0.057). However, DHEA did produce a significant increase in G2 in PC3 cells when
compared to DHA (p<0.01).
Treatment of LNCaP cells with EPA or EPEA at IC50 concentrations did not increase early
or late apoptosis significantly compared to untreated cells (Table 1). However, treatment
with either DHA or DHEA, led to significantly higher levels of LNCaP cells in early
apoptosis (p<0.05, and p<0.01 respectively), when compared to untreated cells
(supplemental figure 1B). DHEA also induced significantly higher apoptosis scores than
DHA (p<0.05) in LNCaP cells, but not late apoptosis. In PC3 cells, neither DHA nor EPA
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induced significant increases in early or late apoptosis compared to untreated cells (Table 1).
Both EPEA and DHEA, however, did elicit significant increases in early apoptosis in PC3
cells compared to untreated cells (p<0.01), and both caused significantly more late-stage
apoptosis than in untreated cells (p<0.05). Treatment of PC3 cells with DHEA showed a
trend towards more apoptosis than with DHA, although this was not statistically significant.
Cannabinoid CB1 and CB2 receptors are expressed in LNCaP and PC3 cells
Western blotting was used to determine the relative expression of CB1 and CB2 receptors in
the two cell lines. The results showed a considerable difference in the relative receptor
distribution/density and form between the two cell lines. CB1 protein was expressed in both
cell lines but the receptor was expressed more highly in LNCaP cells than in PC3 cells,
reaching a borderline significant difference (p=0.057; Figure 2A). CB2 protein was also
expressed in both cell lines, but the antibody detected two prominent bands of different
molecular weight, considered to reflect the presence of two different glycosylation states of
the protein (15). In PC3 cells, the native non-glycosylated form was present in similar
amounts as the glycosylated form (Figure 2B) However, in LNCaP cells, there was lower
expression of the non-glycosylated form, and higher expression of the glycosylated form.
Effects of cannabinoid receptor antagonists on the ability of DHA, EPA and their
ethanolamides to inhibit proliferation of PC3 and LNCaP cells
In LNCaP cells (Figure 2C), combined but not separate administration of the CB1-selective
antagonist , AM281 and CB2-selective antagonist, AM630, significantly reduced the anti-
proliferative effect of EPA, whereas individual antagonists increased the effect, but there
was no effect on EPEA. DHEA was, however, potentiated by AM281 when this was
administered alone or together with AM630, but there was no significant effect on DHA.
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In PC3 cells (Figure 2D), administration of AM281, and AM630, either separately or
together, produced significant inhibition of the anti-proliferative effect of EPEA. Combined
administration of these two antagonists also significantly reduced the anti-proliferative effect
of EPA in this cell line, whereas AM281 or AM630 alone did not affect the potency of this
omega-3 fatty acid. In contrast, separate or combined administration of AM281 and AM630
to PC3 cells did not significantly affect the anti-proliferative potency of DHA and actually
potentiated that of DHEA.
DHEA and EPEA are cannabinoid CB1 and CB2 receptor agonists
DHEA and EPEA each produced a concentration-related displacement of [3H]CP55940 from
specific binding sites in mouse brain and CHO-hCB2 cell membranes (Figures 3A and B
respectively). The apparent mean Ki values of both compounds are shown in Table 2. The
two ethanolamides displaced [3H]CP55940 from brain membranes much more potently in
the presence of the non- selective protease inhibitor, PMSF, than in its absence. This finding
suggests that DHEA and EPEA may be susceptible to metabolism in these membranes, most
likely by FAAH, the enzyme mainly responsible for the metabolism of the endocannabinoid,
anandamide (see introduction). In contrast, in CHO-hCB2 cell membranes, PMSF had very
little (EPEA) or no statistically significant enhancing effect (DHEA), probably reflecting a
lower expression of FAAH in CHO-hCB2 (figure 3C and 3D) cells than mouse brain
membranes (figure 3A and 3B). The binding data we obtained also suggest that DHEA and
EPEA bind to cannabinoid CB1 receptors with higher affinity than to CB2 receptors and that
each of these compounds can fully displace [3H]CP55940 from both these receptors.
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We also found that DHEA and EPEA behaved as CB1 and CB2 receptor agonists, as
indicated by their ability to produce a concentration-related stimulation of [35S]GTPγS
binding to mouse brain (Figure 3E) and CHO-hCB2 cell membranes (Figure 3F). In both
membrane preparations, DHEA displayed higher potency than EPEA. EC50 and Emax values
with 95% confidence limits shown in brackets are listed in Table 2.
FAAH expression and inhibition in LNCaP and PC3 cells
Because of the possibility that DHEA and EPEA may, like anandamide, be metabolized by
FAAH, we went on to determine whether this enzyme is expressed by LNCaP or PC3 cells.
We found that FAAH protein was indeed highly expressed in the androgen receptor positive
LNCaP cells. There was, however, little or no expression of FAAH protein in the androgen
receptor negative PC3 cells (Figure 4A). Since LNCaP cells were found to express FAAH,
we went on to establish whether, as in mouse brain and Chinese hamster ovary membranes,
DHEA or EPEA could be potentiated by inhibiting FAAH in this cell line. We found that
the potency of EPEA was indeed increased by PMSF, as indicated by a significant decrease
in the IC50 in LNCaP cells (Figure 4B), as was that of EPA and DHA. The more selective
FAAH inhibitor, JNJ1661010 (16), increased the antiproliferative potency not only of EPEA
but also of DHEA and EPA, although not of DHA. PMSF did not increase the
antiproliferative potencies of any compound in PC3 cells (Figure 4C).
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Discussion
Our results indicate that the ethanolamide metabolites of two metabolically important
omega-3 fatty acids, EPA and DHA, can activate CB1 and CB2 receptors in PC3 and LNCaP
cells with significant potency. Since it has also been found that these ethanolamides, EPEA
and DHEA, become detectable in vivo after consumption of diets rich in EPA and DHA (4,
17), our results provide the first evidence that EPEA and DHEA may be endocannabinoids.
We also showed that EPEA and DHEA are significantly more potent than their parent fatty
acids at inhibiting prostate cancer cell growth/proliferation. This inhibition appears to result
from changes in both cell cycle arrest and increased apoptosis. However, the precise
mechanisms responsible for this inhibition are not clear at present and appear to differ
between EPEA and DHEA and also between the two prostate cancer cell lines used in this
study.
Although we show a statistically significant difference in potency of the ethanolamides
compared to their fatty acid parent molecules (Figure 1), our data suggests higher IC50 values
than studies have shown for other ethanolamides, such as the omega-6 ethanolamide,
anandamide in prostate cancer cell lines (18). We did not investigate anandamide, and as
this is the first study comparing the IC50 of EPEA and DHEA in prostate cancer cells, we
have no other data to compare with, although our data is consistently reproducible. It is
possible that DHEA and EPEA are less potent than anandamide, as they appear, from our
other data, to also work through CB receptor independent mechanisms. IC50 values for EPA
and DHA in LNCaP cells are similar to those of Chung et al, 2001 (19). The EC50 value of
DHEA for its activation of CB2 receptors was significantly less than its Ki value for the
displacement of the CB1/CB2 receptor ligand, [3H]CP55940, from specific binding sites on
these receptors (Table 2). This was unexpected as DHEA exhibited rather low efficacy as a
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CB2 receptor agonist (Figure 3), suggesting that it is a CB2 receptor partial agonist; a type of
agonist that is not expected to display greater potency in functional than in binding assays.
Clearly, further experiments are required to explain this finding. In contrast, the EC50 value
of EPEA for its activation of brain membrane receptors was significantly greater than its Ki
value for the displacement of [3H]CP55940 from specific binding sites on these receptors.
Furthermore, no statistically significant differences between EC50 and Ki values were
detected for DHEA in brain membrane experiments or for EPEA in experiments with CHO-
hCB2 cell membranes, when the binding assays were performed in the presence of PMSF,
suggesting that inhibition of FAAH and/or increased substrate availability eliminated any
differences. PMSF was included in our [3H]CP55940 binding experiments to prevent the
metabolism of EPEA or DHEA by the fatty acid ethanolamide-metabolizing enzyme,
FAAH. The presence of PMSF in the [35S]GTPγS binding assays was deemed unnecessary
as the concentration of protein, and hence of FAAH, was much less in these assays than in
the ligand displacement assays.
In LNCaP cells, the significant decrease in S phase and in G1 arrest elicited by DHA and
DHEA, respectively, was in marked contrast to the lack of any effect of EPA or EPEA on
any of the cell cycle parameters, compared to untreated cells. In PC3 cells, however, EPA
did cause significant cell cycle arrest in the G1 phase (increase in G1 cells) compared to
untreated cells, and DHA induced a marked decrease in S phase cells, suggesting both these
fatty acids were able to reduce proliferation in the PC3 cells but only DHA was effective in
the LNCaP cells, perhaps indicating a possible role for wild-type p53, since PC3 cells have a
mutant p53 protein. These observations are in contrast to data obtained in some previous
investigations in which the effects of different synthetic cannabinoid receptor agonists on
cell cycle phases were explored. For example, Sarfaraz et al. (2006) demonstrated G1 arrest
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with the CB1/CB2 receptor agonist, R-(+)-WIN55212 (20) . They are, however, in agreement
with other studies that demonstrated a decreased G1 and increased G2 after treatment with
R-(+)-methanandamide, a CB1-selective anandamide analogue and JWH-015, a CB2-
selective agonist .This suggests that alternative mechanisms may account (21) for the effects
of different natural and synthetic cannabinoids on cell cycle parameters. Indeed, in our
studies, we have demonstrated differential effects between fatty acids and their
ethanolamides on the cell cycle, which differed somewhat according to the cell line, but
which still resulted in significant changes in tumour cell proliferation/growth. Why DHEA
and EPEA should have such different effects on the cells is currently unclear. One
possibility is that it reflects differences between the pharmacological properties of these
ethanolamides or indeed of their respective metabolites and the possible presence of other
receptors.
We found that , whereas DHEA could induce significant apoptosis in both LNCaP and PC3
cells, only EPEA increased apoptosis in PC3 cells, a further indication of differences
between the two ethanolamides. Since PC3 cells, but not LNCaP cells, express the pro-
apoptotic p53 oncogene mainly as the inactive, mutant form, our data suggest that the EPEA
or DHEA can activate apoptosis through either p53 dependent or p53 independent
mechanisms in different cell lines (22). This is interesting, as previous findings from our
group showed that omega-3 LCPUFA could induce apoptosis, both dependently and
independently of p53 activation, and could actually alter the expression of mutant-p53 to
reactivate/ re-establish wild-type function in breast cancer cells (23). It remains to be
determined if the omega-3 ethanolamides can influence apoptosis through a similar
reactivation mechanism in either breast or prostate cancer cells. It is also interesting to note
that despite multiple repetition, we continued to find variable results with using the EPA and
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DHA fatty acids alone, particularly with regards to early apoptosis, and only in PC3 cells.
Perhaps the fatty acids interrupt the fluidity of PC3 more than LNCaP cell membrane, and
interfere with annexin binding to phosphatidyl serine on cell membranes, or affect
membrane fluidity, giving spurious results. Other studies, however, have not discussed
such a phenomenon with PC3 cells, and it may be a unique effect with our particular cell
line? What is apparent from our observations is that the omega-3 ethanolamides generally
affect cell cycle functions and apoptosis with greater potency than their parent fatty acids.
This could form the basis for the development of novel therapeutic agents in cancer.
We also explored the possibility that either or both EPEA and DHEA might act through
cannabinoid CB1 and /or CB2 receptors to induce their inhibitory effects on the proliferation
of LNCaP and PC3 prostate cancer cells. It is already known that anandamide, an
endogenous omega-6 ethanolamide, can both activate cannabinoid receptors and inhibit
cancer cell proliferation (24, 25). Previous studies have shown that prostate cancer cells
express cannabinoid receptors (18, 26) and that LNCaP and PC3 cells express both CB1 and
CB2 receptors (27). We confirmed this in our cell lines, and then showed that EPEA and
DHEA displayed significant potency in vitro as CB1 and CB2 receptor agonists. However,
we also obtained evidence that the anti-proliferative effects of EPEA in LNCaP cells and of
DHEA in LNCaP and PC3 cells are not CB1 or CB2 receptor-mediated. This was deduced
from data obtained in experiments with the CB1-selective antagonist, AM281, and the CB2-
selective antagonist, AM630, each applied at a concentration (1 µM) that has been used in
other investigations to identify effects that are CB1 and/or CB2 receptor-mediated (28, 29,
30). Thus, we found that separate or combined administration of these antagonists had no
effect on the anti-proliferative potencies of EPEA or DHA in LNCaP cells or of DHA in
PC3 cells and increased rather than decreased the potency of DHEA in both cell lines
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(Figure 1). These findings are not altogether unexpected as it has been reported that
anandamide-induced anti-proliferative effects in some cancer cell lines can be potentiated by
CB1 or CB2-selective antagonists (24, 25). In contrast, we did find that the anti-proliferative
potencies of EPEA in PC3 cells and of EPA in PC3 and LNCaP cells were reduced by
AM281 and AM630 when these antagonists were administered separately (EPEA) or in
combination (EPEA and EPA). Even so, it is currently unclear whether EPA, possibly after
its conversion to EPEA, or direct administration of EPEA were indeed acting through
cannabinoid receptors to inhibit PC3 cell proliferation, as EPEA produced this effect with a
potency well below the potency it displayed as a CB1 or CB2 receptor agonist (Figure1 and
Table 1).
Further research is now needed to establish whether, as has been proposed for anandamide
(23, 24), the DHEA effect was potentiated by AM281 and AM630 in our experiments
because blockade of cannabinoid receptors increased its ability to inhibit cancer cell
proliferation through one or more cannabinoid receptor-independent mechanisms in the
cancer cell lines we used. It will be important, therefore, to establish the extent to which
DHEA targets non-CB1, non-CB2 receptors, particularly transient receptor potential V1
(TRPV1) cation channels. These channels can be activated by both anandamide and omega-
3 polyunsaturated fatty acids and Maccarrone and colleagues have demonstrated that
cannabinoid receptor activation can prevent apparent TRPV1-mediated apoptosis induced by
anandamide (24, 31). Consequently, it is possible that by blocking cannabinoid receptors,
we reduced cannabinoid receptor-mediated protection of LNCaP and PC3 cells, thereby
increasing the ability of DHEA to induce apoptosis through vanilloid receptors, which are
expressed in both these cell lines(32). Interestingly, whereas DHA is a potent TRPV1
agonist, EPA inhibits the activation of this cation channel by various agonists (33). Whether
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DHEA and EPEA display this same difference in their pharmacology remains to be
established. Since recent studies have demonstrated that CB1 and CB2 receptors do not
mediate apoptosis in malignant astrocytomas if they are coupled to the prosurvival signal
AKT (34), further research is also needed to establish the extent to which cannabinoid
receptors couple to AKT in our cancer cell lines.
Our data suggest that EPEA and DHEA resemble the endocannabinoid, anandamide, not
only in their ability to activate CB1 and CB2 receptors, but also in their susceptibility to
metabolism by FAAH. Thus, the potency with which each of these omega-3 ethanolamides
displaced [3H]CP55940 from brain membranes was increased by the FAAH inhibitor,
PMSF, as indeed was the potency of anandamide in this binding assay (data not shown).
Moreover, PMSF increased the anti-proliferative potency of EPEA, as well as of EPA and
DHA (but not DHEA), in LNCaP cells in which we found FAAH protein to be highly
expressed. Although PMSF is not a selective inhibitor of FAAH, it is likely that it did
produce this potentiation by inhibiting this enzyme. This is supported by the observations
that, firstly, PMSF did not increase the anti-proliferative potencies of EPEA, DHEA, EPA or
DHA in PC3 cells in which we detected little or no expression of FAAH protein, and
secondly, a more selective FAAH inhibitor, JNJ1661010, also increased the anti-
proliferative potencies of EPEA and EPA in LNCaP cells (16). Moreover, in contrast to
PMSF, JNJ1661010 also increased the anti-proliferative potency of DHEA in LNCaP cells,
though it failed to affect the potency of DHA. The observation that inhibition of FAAH in a
prostate cancer cell line that expresses this enzyme could decrease the IC50 values of EPA
and DHA, supports the hypothesis that some of the many known effects of these omega-3
fatty acids, may actually arise as a result of their conversion in situ to their endocannabinoid
metabolites in some cells, a process which has been shown to occur in some animal
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tissues(4, 35). Our finding that PC3 cells do not seem to express FAAH conflicts with a
previous report, however, this could be due to inherent differences in cell lines between
different laboratories, or to differential limits of detection by different antibodies/techniques
(36). In our study, PC3 cells showed, in some replicates, a barely detectable level of
expression, and in other replicates, no expression at all, whereas it was easily detected in
LNCaP cells.
In conclusion, we show here for the first time, that the omega-3 ethanolamides, EPEA and
DHEA, are more potent than their parent fatty acids, EPA and DHA, at inhibiting prostate
cancer cell proliferation, and that the anti-proliferative effects they produce appear to have
different underlying mechanisms and may be cell specific. We also demonstrate that these
ethanolamides activate CB1 and CB2 receptors with significant potency and that the
potencies of both can be enhanced by inhibiting the anandamide-metabolizing enzyme,
FAAH, both in brain tissue and in FAAH-expressing cancer cells. We propose, therefore,
that EPEA and DHEA should be classified as endocannabinoids It has been shown
previously that these omega-3 ethanolamides are generated in vivo after consumption of
their parent fatty acids, EPA and DHA a finding that may explain some of the anti-tumour
effects of these omega-3 fatty acids that have been observed in vivo in other studies (17, 37,
38). The enhancing effect of FAAH inhibition in FAAH-expressing LNCaP cells on the
anti-proliferative effects of EPA and DHA also suggests, for the first time, that these cells
can convert these fatty acids to ethanolamides in situ. It is also noteworthy that the results
we obtained from our experiments with LNCaP and PC3 cells suggest that the omega-3
ethanolamides, EPEA and DHEA have different, albeit overlapping, pharmacological
fingerprints, as has been shown for other cannabinoid receptor ligands (39). By identifying
the particular pharmacological targets and physiological mechanisms through which EPEA
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and DHEA induce their inhibition of prostate cancer cell proliferation, it may be possible to
identify tumours which are more liable to respond to treatment with either omega-3 fatty
acids or their ethanolamides, perhaps in conjunction with a FAAH inhibitor or even with
cannabinoid CB1 and/or CB2 receptor antagonists.
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Funding
This work was supported by grants from the National Institutes of Health (DA-009789),
TENOVUS Scotland, and NHS Grampian Endownments fund.
Acknowledgements The authors wish to thank Mrs Lesley A. Stevenson for technical support, and the Aberdeen
University Flow Cytometry facility.
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Legends to figures
Figure 1: Structure of eicosapentaenoic and docosahexaenoic acid ethanolamides and their
effects on cell viability. A) Chemical structure of EPEA and DHEA compared to
anandamide. B) IC50 values as determined from MTT assays in LNCaP and C) PC3 cells
treated with either fatty acids (EPA and DHA), or ethanolamides (EPEA or DHEA) for 24
hours. Error bars represent standard error of mean. *p=<0.05, †p=<0.01 (n=3).
Figure 2: A-B; Cannabinoid receptor expression. A graphical representation of expression
as a percentage relative to β-actin internal control and also a representative example of
western blotting. A) CB1 receptor protein expression in PC3 (PC) and LNCaP (LN) cells. β-
actin used as loading control and positive control is rat cellebellum lysate (+). B) CB2
expression in PC3 (PC) and LNCaP (LN) cells. β-actin used as loading control and positive
control is HL-60 cells (+). Error bars represent standard error of mean (n=3). C-D; Effect of
inhibiting cannabinoid receptors in C) LNCaP cells and D) PC3 cells. IC50 values as
determined from MTT assays. (AM281, a CB1 selective antagonist; AM630, a CB2-selective
antagonist; Mix = mixture of both antagonists; FA = fatty acid; EA = ethanolamide).
AM251 and AM630 were each administered at a concentration of 1 µM. Error bars represent
standard error of mean. *p=<0.05, †p=<0.01, comparing cells treated with a fatty acid or
ethanolamide alone and cells also treated with AM281 or AM630 or with both AM281 and
AM630 (Mix), (n=3-5).
Figure 3: Functional activity of ethanolamides. A-D) Displacement of [3H]CP55940 by A)
EPEA and B) DHEA from specific binding sites on MF1 mouse whole brain membranes
(n=4), and displacement of [3H]CP55940 by C) EPEA and D) DHEA from specific binding
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sites on CHO-hCB2 cell membranes (n=8). Each experiment has been performed in the
absence and presence of 100 μM PMSF. Each symbol represents the mean percent
displacement ± SEM. Mean Ki values with 95% confidence limits shown in brackets are
shown in Table 2. E-F) Mean log concentration-response curves of EPEA and DHEA. Each
symbol represents the mean percentage change in binding of [35S]GTPγS to E) MF1 mouse
brain and F) CHO-hCB2 cell (B) membranes ± SEM. Mean EC50 and Emax values are shown
in Table 2.
Figure 4: Role of FAAH in LNCaP and PC3 cells. A) A representative example of western
blotting. β-actin used as internal loading control. Positive control (+) is UM-84 cell line.
Combined protein expression data shown as bar chart indicating ratio of expression of
protein of interest to β-actin ± SEM. B) Effect of inhibiting FAAH with PMSF or
JNJ101660 (JNJ), in LNCaP, cells and C) inhibition with PMSF in PC3 cells. (PMSF =
phenylmethylsulfonyl fluoride, JNJ = selective FAAH inhibitor, JNJ10660. FA=fatty acid,
Eth=ethanolamide). Error bars represent standard error of mean. *p=<0.05, †p=<0.01,
comparing cells treated with either fatty acid only, or ethanolamide only, against those
treated with FAAH inhibitors.
Table 1: Effects of ethanolamides on cell cycle and apoptosis.
Table 2: [3H]CP55940 and [35S]GTPγS binding assays
Supplementary figure 1: Example of ethanolamide effects on cell cycle and apoptosis A)
Cell cycle analysis by DNA propidium iodide (PI) staining showing a decrease in G1 phase
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of cell cycle with EPEA (n=4) and B) Density plots of PI (y-axis) and Annexin (x-axis)
staining to indicate increase in apoptotic cells (lower right quadrant) after treatment with
DHEA (n=3).
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Table 1: Effects of ethanolamides on cell cycle and apoptosis
Cell line Untreated cells EPA IC50
EPEA IC50 DHA IC50 DHEA IC50
LNCaP Cell CycleG1 76.13 ±4.15 72.42 ±1.57 78.84 ±2.03 76.65 ±3.89 66.83 ±0.77*/**S 12.55 ±2.63 10.40 ±0.914 9.86 ±1.74 7.45 ±1.53* 9.63 ±1.88
G2 12.52 ±1.67 9.57 ±0.88 10.79 ±1.17 8.21 ±0.90* 10.07 ±1.23 PC3 G1 41.67 ±4.61 62.26 ±2.37† 45.18 ±4.31 52.04 ±6.67 43.49 ±1.27
S 21.69 ±5.02 6.12 ±1.45† 16.69 ±3.48 19.88 ±7.48 13.65 ±4.51G2 35.04 ±0.55 28.75 ±2.03 36.45 ±7.40 27.73 ±0.98† 41.81 ±4.54††
ApoptosisLNCaP Early 10.94 ±2.43 15.30 ±3.66 16.19 ±5.21 19.76 ±3.71* 43.81 ±4.70†
Late 14.94 ±3.31 19.44 ±10.99 17.01 ±5.48 18.52 ±5.86 29.59 ±10.94PC3 Early 9.47 ±2.40 20.09 ±10.57 31.63 ±7.10* 20.41 ±17.01 28.75 ±0.10*
Late 5.84 ±0.41 10.08 ±6.44 27.52 ±13.33 8.79 ±4.41 24.36 ±10.31**p<0.05, †P<0.01 against untreated cells, **p<0.05, ††p<0.01 between fatty acid and corresponding ethanolamide. Cells treated with
IC50 concentrations (see Figure 1) of each compound for 24 h. All experiments repeated 3 times.
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Table 2:
Compound
aCB1$Ki (nM)
No PMSF(n=4)
aCB1$Ki (nM)
with PMSF(n=4)
bCB2$Ki (nM)
No PMSF(n=8)
bCB2$Ki (nM)
with PMSF(n=8)
aCB1
*EC50 (nM)(n=4)
aCB1
*Emax
bCB2
*EC50 (nM)(n=4)
bCB2
*Emax
EPEA 2000(989 & 4045)
55(37.5 & 80.9)
9027(6451 & 12630)
3440(2324 & 5092)
1361(223 & 8288)
40.6(24.7 & 56.6)
397.1(22.2 & 7085)
22.9(13.4 & 32.5)
DHEA 633(292 & 1372)
124(94.8 & 162)
3843(2995 & 4932)
2141(1320 & 3473)
50(2.74 & 911)
26.5(16.3 & 36.8)
42(2.6 & 676)
23.1(15.7 & 30.5)
aCB1 receptors have been investigated using MF1 mouse whole brain membranesbCB2 receptors have been investigated using CHO-hCB2 cell membranes$Ki values have been calculated for displacement of [3H]CP55940*EC50 and Emax values have been calculated for stimulation of [35S]GTPγS binding
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